The invention relates to an apparatus for liquid disposal and distribution for use in an automatic culture system which performs an automatic culturing of biological tissues and cells, and more particularly to such apparatus for transferring or distributing cells being cultured from a culture vessel into a centrifuge tube or vice versa and for disposing unnecessary liquid from the culturing vessel.
The technique of culturing biological tissues and cells represents an essential and fundamental experimental process in the study of cells in various fields including the medical, biological, pharmaceutical and agricultural fields. However, the culture of biological tissues and cells over successive generations involves a technical difficulty, which prevents a stable strain being cultured from being obtained in practice. Thus, there has been a need to establish a procedure for culturing biological tissues and cells which makes it possible to obtain a stabilized strain. Recently, a culture technique in a gaseous environment maintained within an incubator has been developed, and has enabled the culturing over successive generations of cells of a particular variety such as those of liver, neuron, pituitary gland, which have been heretofore considered difficult to be cultured.
The culture over successive generations of the kind described will be briefly summarized below. A given number of cells which are to be cultured over successive generations are diluted in a culture solution in the form of a suspension, which is then injected into a culture vessel such as petri dish. The vessel is placed still in an incubator for culturing of the cells under a given atmosphere. After a given period of time, the vessel is removed from the incubator and the growth of cells examined under a microscope. When it is determined that the intended growth of cells has extended to the full extent of the vessel, they are transferred to a strain-free clean bench, and the culture solution in the vessel is withdrawn with a pipette and disposed. Subsequently, a buffer solution is injected into the vessel to clean the remaining cells, and then withdrawn for disposal with a pipette. To render the grown cells which attach to the bottom of the vessel almost freely releasable therefrom, an enzyme solution such as tripsin is injected into the vessel and the latter left intact for a given period. After such period, the enzyme solution is withdrawn from the vessel with a pipette and disposed, and a culture solution is again injected into the vessel. The culture solution is repeatedly withdrawn and discharged through a pipette to cause an oscillation and agitation which enables the grown cells to be completely released from the bottom of the vessel into a suspension in the culture solution. The cells in suspension are transferred into a centrifuge tube with a pipette, and placed in a centrifuge to separate the cells from the solution. Thereupon, the cells remain attached to the bottom of the tube, while the culture solution will be a supernatant solution, which is disposed by tilting the tube. A culture solution is again injected into the centrifuge tube and is agitated by utilizing the withdrawal and discharge operation with a pipette in order to separate the cells from each other so that they are uniformly suspended in the culture solution within the centrifuge tube. Finally, the solution is distributed into a pair of culture vessels in an equal amount to complete one culture operation.
It will be seen that with the culture technique described, it is necessary to remove the culture vessel from the incubator and place it in the outer atmosphere in order to examine the growth of the tissues or cells under the microscope. This causes a sudden change in the culturing condition since the cells or tissues are placed out of the given environment maintained within the incubator including a given gas atmosphere, temperature and humidity. This causes a delicate influence upon the tissues or cells being cultured and also involves an unavoidable contamination by miscellaneous strains present in the atmosphere.
In addition, the various operations required for culturing over successive generations which should take place on the basis of the observation under the microscope, depend on a manual operation by an operator on the clean bench. This means that any slight difference in the various operations which occur from operator to operator may have a direct influence upon the culturing result of the tissues or cells since the experience and skill of culturing technique varies from operator to operator. Thus, it is difficult to provide a standard procedure for the culturing technique which makes it impossible to obtain cultured tissues or cells of uniform quality. As a consequence of this, different groups of researchers, conducting a common study on the same theme, may reach different conclusions, depending on the quality of the tissues being cultured. In extreme cases, the conclusions may be opposite to each other. Thus it will be seen that the reliability cannot be expected when the tissues or cells are cultured with the conventional technique.
It is generally accepted that the training of a skilled operator requires at least two years. As a result, there has been a continued shortage of skilled operators. It has therefore become necessary for the researchers to perform the culturing operation themselves rather than devotedly directing their efforts to their study.
In view of these considerations, the present invention is directed towards an automatic culture system capable of performing the described culturing operations automatically in order to eliminate the contamination which may occur as a result of the exposure of the tissues or cells being cultured to the atmosphere and to eliminate the influence of manual operations upon the cultured results, thereby permitting a standard and uniform procedure to be adopted for conducting the various culturing operations.
As will be appreciated from the foregoing description, this automatic culture apparatus is able to withdraw and dispose from the culture vessel a liquid such as culturing, buffer and enzyme solution, to render grown cells in the culture vessel as a suspension in a fresh culturing solution for distribution into the centrifuge tube. The apparatus is further able to separate the centrifuged cells from each other for suspension in a fresh culturing solution in order to permit their distribution into a pair of fresh culture vessels in an equal amount. In the present apparatus, these operations are performed by a fixed displacement pump of a syringe type which is connected with a pipette for conducting a withdrawal and discharge operation. However, it will be appreciated that when such pipette is used, the admixture of a liquid from a previous step with a fresh solution delivered into the culture vessel or centrifuge tube during the next following step must be avoided since otherwise there occur adverse influences upon the culturing result. Hence, the fresh one must be used for each operation. Furthermore, as mentioned previously, before the grown cells are transferred into the centrifuge tube, the culturing solution within the vessel is repeatedly withdrawn and discharged with a pipette to cause an agitation of the cells so that they are completely released from the bottom of the vessel and rendered as a suspension in the culturing solution. It is desirable that such agitation be achieved with a high efficiency.